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doi:10.1016/S0022-2836(02)01232-9
J. Mol. Biol. (2003) 325, 325–335
Rabbit Immune Repertoires as Sources for
Therapeutic Monoclonal Antibodies: The Impact of
Kappa Allotype-correlated Variation in Cysteine
Content on Antibody Libraries Selected by
Phage Display
Mikhail Popkov1, Rose G. Mage2*, Cornelius B. Alexander2
Sujatha Thundivalappil1, Carlos F. Barbas III1* and Christoph Rader1*
1
Department of Molecular
Biology and The Skaggs
Institute for Chemical Biology
The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, CA 92037, USA
2
Laboratory of Immunology
National Institute of Allergy
and Infectious Diseases
National Institutes of Health
Bethesda, MD 20892, USA
The rabbit immune repertoire has long been a rich source of diagnostic
polyclonal antibodies. Now it also holds great promise as a source of
therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal
antibody. The allotypic diversity of rabbit immunoglobulins prompted us
to compare different rabbit immune repertoires for the generation and
humanization of monoclonal antibodies that bind with strong affinity to
antigens involved in tumor angiogenesis. In particular, we evaluated the
diversity of unselected and selected chimeric rabbit/human Fab libraries
that were derived from different kappa light chain allotypes. Most rabbit
light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared
with mouse and human kappa light chains. Here we evaluate the impact
of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits
with mutant bas and wild-type parental b9 allotypes are excellent sources
for therapeutic monoclonal antibodies. Featured among the selected
clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1 nM to both human and mouse Tie-2, which will
facilitate its evaluation in mouse models of human cancer. Examination
of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study
revealed that rabbits exhibit an HCDR3 length distribution more closely
related to human antibodies than mouse antibodies.
q 2003 Elsevier Science Ltd. All rights reserved
*Corresponding authors
Keywords: rabbit monoclonal antibodies; antibody engineering; antibody
library; phage display; immunoglobulin kappa light chain
Introduction
Monoclonal antibodies (mAbs) are gaining
importance in the treatment of various conditions,
Abbreviations used: mAb, monoclonal antibody; CDR,
complementarity determining region; HCDR3, heavy
chain complementarity determining region 3; LCDR3,
light chain complementarity determining region 3; V,
variable domain; C, constant domain; NZW, New
Zealand White; ang-2, angiopoietin-2.
E-mail addresses of the corresponding authors:
[email protected]; [email protected];
[email protected]
including cancer, cardiovascular diseases, and
viral infections. In concert with their clinical acceptance, mAbs have become commercially viable
drugs.1,2 Today, mAbs are generated by either
hybridoma technology or from antibody libraries.3
The development of antibody libraries has been
greatly influenced by the development of display
technologies and vice versa. The physical connection of antibody phenotype (protein) and genotype
(gene) effectively allows selection rather than
screening of antibody libraries. The integration of
antibody libraries and phage display technology4
about a decade ago5,6 was a key event in this
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
326
respect. More recently, display technologies other
than phage display have been applied to antibody
libraries, including ribosome, yeast, and bacterial
display.7,8
Whereas the hybridoma technology9 is practically confined to rodents (mice, rats, and hamsters), antibody libraries allow the generation of
mAbs from virtually any species whose immunoglobulin genes are known.10 In addition, antibody
libraries have been used to exploit large naı̈ve and
synthetic antibody repertoires, or combinations of
both, for the generation of human mAbs.11,12 In
contrast to antibodies derived from large naive or
synthetic repertoires, however, antibodies from
immune animals are subjected to in vivo selection
and, thus, are more likely to recognize a given antigen selectively, that is, with less cross-reactivity to
other antigens. It is conceivable that the ability to
generate mAbs from a variety of species will be
important for the identification of highly conserved
human antigens or highly conserved epitopes of
human antigens. The epitope repertoire of a given
human antigen recognized by non-human antibodies is different for each species. As a result, epitopes that are not immunogenic in mice might be
immunogenic in other species, for example in
rabbits. Highly conserved epitopes often display
functional binding sites. The generation of mAbs
against functional binding sites, that is, the generation of mAbs that agonize or antagonize
functional interactions, is relevant for therapeutic
applications.13
The rabbit antibody repertoire, which in the
form of polyclonal antibodies has been used in
diagnostic applications for decades, is an attractive
alternative to the mouse antibody repertoire for the
generation of mAbs to human antigens. Importantly, rabbits are evolutionarily distant from mice.
Rabbits belong to the family of Leporidae, which
is not part of the large and diverse group of
rodents. As we have demonstrated recently, rabbit
mAbs selected from antibody libraries by phage
display can be humanized while retaining both
high specificity and strong affinity to the human
antigen.14,15 Rabbit mAbs that cross-react with
human and mouse antigens are of particular
relevance for the preclinical evaluation of therapeutic antibodies in mouse models of human
diseases. In contrast, humanized and human antibodies that are derived from immune mice, either
indirectly through humanization or directly
through transgenic mice containing human
immunoglobulin loci,16 are negatively selected
against epitopes displayed by the mouse antigen
and, thus, often lack cross-reactivity. In fact, the
lack of cross-reactivity with the mouse antigen of
humanized mouse mAbs directed to human
integrin avb3, human VEGFR-2, and human VEGF
has been a major difficulty in their preclinical
development.17
Here, we evaluate different rabbit immune
repertoires as sources for therapeutic mAbs. In particular, rabbit immune repertoires were compared
Rabbit Antibody Libraries
with respect to the generation and selection of
phage libraries displaying chimeric rabbit/human
Fab, a relevant format for the humanization of
rabbit mAbs.14,15 Further we have analyzed CDR3
length distributions for both light and heavy chains
in view of the fact that these regions are conserved
in the process of antibody humanization.
The antibody diversity generated by VHDJH
rearrangements in rabbits is more limited than in
mice and humans, because one out of . 50 functional VH gene segments, VH1, is predominantly
used. Much of the diversity in rearranged VH1DJH
genes develops by somatic gene conversion-like
and somatic hypermutation mechanisms.18,19 In
contrast to the limited VHDJH rearrangements, VkJk
rearrangements in rabbits are much more diverse
and, thus, the resulting rearranged kappa light
chain genes may compensate for the limited diversity of rearranged heavy chain genes.20 Like the
heavy chain genes, kappa light chain genes are
further diversified by somatic gene conversionlike as well as by somatic hypermutation
mechanisms.21 Thus, the kappa light chain appears
to be a major contributor toward generation of the
antibody diversity of the rabbit immune repertoire.
In contrast to mice and humans, which have only
one kappa light chain isotype, rabbits have two,
K1 and K2,22,23 including highly diverse allelic
variants of Kl (b4, b5, b6, and b9 allotypes). In normal rabbits, , 70 –90% of the serum antibodies are
of the Kl isotype, while the remainder consists of
both lambda light chains and kappa light chains
of the K2 isotype.18 By contrast, antibodies in the
serum of rabbits from the Basilea strain,24 which
does not express kappa light chains of the K1 isotype due to a splice site mutation,25 are predominantly composed of lambda light chains and
kappa light chains of the K2 isotype (bas allotype).
Most rabbit kappa light chains of the K1 isotype
have an unusual disulfide bridge that joins the
variable and constant domains, usually through
cysteine residues at positions 80 and 171.26 This
disulfide bridge links framework region 3 of the
variable kappa light chain domain with the constant kappa light chain domain, a linkage not seen
in mouse or human antibodies (Figure 1). Exceptions are Basilea mutant rabbits that cannot express
the K1 isotype and rabbits of the wild-type parental b9 allotype where a cysteine at position 108
in framework region 4 of the variable kappa light
chain domain can substitute for cysteine 80.
The genes of bas and b9 wild-type rabbits are
evolutionarily close and more frequently express
kappa light chains that do not encode a cysteine
80. This is of particular interest for the generation
of chimeric antibodies consisting of rabbit variable
and human constant domains. The fusion of rabbit
variable kappa light chain domains containing
cysteine 80 to the human kappa light chain constant domain, which does not provide a matching
cysteine, will result in a free thiol group, which is
likely to be disadvantageous for the expression of
antibody fragments in Escherichia coli27 and, thus,
327
Rabbit Antibody Libraries
Figure 1. Intrachain disulfide bridges in the kappa
light chain. Shown is the alpha carbon backbone in the
crystal structure of the kappa light chain (Vk-Ck) of
humanized Fab D3h44 (PDBID 1JPT)37 with the intrachain disulfide bridges of the variable (Cys23-Cys88)
and the constant domain (Cys134-Cys194). Most rabbit
kappa light chains have an additional intrachain disulfide bridge that joins the variable and constant domains,
usually through cysteine residues at positions 80 and
171 or, in the b9 allotype, through cysteine residues at
positions 108 and 171. Although this linkage is not seen
in mouse or human antibodies, the distances between
the amino acids that can be cysteine in rabbit kappa
light chains would allow for disulfide bridge formation.
The calculated distances in D3h44 are 6.7 Å for Cys23Cys88, 6.5 Å for Cys134-Cys194, 6.2 Å for Pro80-Ser171,
and 7.0 Å for Arg108-Ser171. This Figure was prepared
with ViewerLite 4.2 software (Accelrys).
disadvantageous for the selection of antibody fragments from phage display libraries. In addition, a
free thiol group in antibody fragments might have
a negative impact on their stability in vitro (storage)
and in vivo (application). An indication that
cysteine 80 may restrict the selectable diversity of
chimeric rabbit/human Fab libraries came from
our previous study, in which all three chimeric rabbit/human Fab that we selected against human
A33 antigen contained rabbit variable kappa light
chains without cysteine 80.14 By contrast, one chimeric rabbit/human Fab that we selected against
human CCR5 receptor contained a rabbit variable
kappa light chain with cysteine 80.15 This cysteine
was eliminated during the humanization process.
One goal of the present study was to determine
whether rabbit immune repertoires with higher
expression of kappa light chains that do not encode
cysteine 80 are a superior source in terms of selectable diversity.
Results and Discussion
Immunization
Six rabbits, two each homozygous for the b4, bas,
and b9 kappa allotypes, were immunized with an
equimolar mixture of two interacting proteins, a
recombinant human Tie-2/Fc fusion protein and
recombinant human angiopoietin-2 (ang-2). We
chose this immunogen for two reasons. First, both
the endothelial cell receptor tyrosine kinase Tie-2
and its ligand ang-2 are involved in angiogenesis.28
Angiogenesis is fundamental to pathologic processes such as diabetic retinopathy, rheumatoid
arthritis, and cancer.29 Tie-2, ang-2, and Tie-2/ang2 complex are potential targets for antiangiogenic
therapy and, thus, antibodies with these specificities are therapeutically relevant. Second, since the
main goal of this study was to compare the
different rabbit immune repertoires in terms of
selectable diversity, it was hoped that the use of a
mixture of two interacting proteins as immunogen
would contribute to the diversity of the humoral
immune response.
Library generation
Analyses of the sera from all six immunized rabbits by ELISA showed that the immunization
resulted in a strong immune response against both
the Tie-2 and ang-2 proteins of the complexed
immunogen. At a dilution of 1:1000, the absorbance at 405 nm was . 2.0 for all antisera and
, 0.15 for all preimmune sera when using 100 ng
coated antigen and a 1:500 dilution of horseradish
peroxidase-conjugated goat anti-rabbit Fc polyclonal antibodies for detection. Chimeric rabbit/
human Fab libraries in phagemid vector pComb3X
were generated from cDNA derived from spleen
and bone marrow RNA of the immune rabbits as
described14,30 (Figure 2). All rabbits and tissues
were handled separately, resulting in 12 independent libraries with each of the three immune
Figure 2. Generation of chimeric rabbit/human Fab.
Schematically depicted is the amplification and assembly
of the Fab building blocks. The light chain comprises a
rabbit variable (kappa or lambda) light chain domain
(VL) and a human constant kappa light chain domain
(Ck). The heavy chain fragment comprises a rabbit
variable heavy chain domain (VH) and a human constant
heavy chain domain (CH1). Primers for amplification and
assembly of these building blocks are shown as arrows.
The assembled dicistronic expression cassette is ligated
into phagemid vector pComb3X through asymmetric
Sfi I sites.
328
Rabbit Antibody Libraries
Table 1. Origin and number of independent transformants of chimeric rabbit/human Fab libraries
Library
Spleen 1
Spleen 2
Bone marrow 1
Bone marrow 2
Total
NZW
Basilea mutant
b9 wild-type
3.2 £ 108
4.95 £ 108
2 £ 108
2.74 £ 108
2.65 £ 108
4.6 £ 108
0.9 £ 108
1.5 £ 108
0.44 £ 108
0.76 £ 108
1.7 £ 108
3.4 £ 108
0.76 £ 109
1.08 £ 109
1.04 £ 109
repertoires represented by a complexity of approximately 1 £ 109 independent transformants (Table 1).
Library diversity
Randomly picked independent transformants
from each of the 12 unselected libraries were
analyzed for protein expression and DNA
sequence. The expression of chimeric rabbit/
human Fab was analyzed by ELISA using goat
anti-human Fab and goat anti-human kappa light
chain polyclonal antibodies for capture and a rat
anti-HA mAb conjugated to horseradish peroxidase for detection. The Fd fragment of Fab
expressed by phagemid vector pComb3X contains
a C-terminal HA tag.30 Positive signals were
obtained for 89% (71/80), 90% (77/86), and 93%
(80/86) of clones derived from New Zealand
White (NZW), Basilea mutant, and b9 wild-type
rabbits, respectively. All positive clones were subsequently analyzed by DNA sequencing of both
rabbit VH and VL coding regions, revealing 100%
unique sequences. (The GenBank accession numbers are AY171619-AY171761 (NZW), AY171762AY171914 (Basilea mutant) and AY175467AY175626 (b9 wild-type).) The deduced heavy
chain complementarity determining region 3
(HCDR3) and light chain complementarity determining region 3 (LCDR3) length diversity ranged
from 3 to 22 and 8 to 14 amino acid residues,
respectively, and revealed a similar distribution in
all three immune repertoires although a bias
Figure 3. CDR3 length diversity
of chimeric rabbit/human Fab
libraries. The CDR3 length in number of amino acids (x axis) was
defined based on the Kabat numbering scheme as the number of
codons following the conserved
Cys88 for LCDR3 or the conserved
Arg94 for HCDR3 up to the conserved Phe98 for LCDR3 or up to
the conserved Trp103 for HCDR3.
329
Rabbit Antibody Libraries
Table 2. Length distribution of HCDR3 amino acid sequences in rabbit, human, and mouse
Species
Number
Range
Median
Mean ^ SD
Skewnessa
Kurtosisb
Rabbit
Humanc
Mousec
228
177
1004
3–22
2–26
2–19
12
13
9
11.6 ^ 3.1
13.1 ^ 4.4
9.4 ^ 2.8
0.3
0.5
20.005
1.2
0.5
0.04
a
Skewness characterizes the degree of asymmetry of a distribution around its mean. A normal distribution is symmetric
(skewness ¼ 0).
b
Kurtosis characterizes the relative peakedness or flatness of a distribution compared with the normal distribution (kurtosis ¼ 0).
c
Calculations were based on published data.32
toward an LCDR3 length of ten amino acid residues was noted for Basilea mutant and b9 wildtype but not NZW rabbits (Figure 3). The arithmetic mean ^ SD LCDR3 length was 11.0(^ 1.6)
for NZW rabbits (n ¼ 71), 10.0(^ 0.9) for Basilea
mutant rabbits (n ¼ 77), and 10.6(^ 1.2) for b9
wild-type rabbits (n ¼ 80). Thus, in agreement
with earlier studies,20 our large sample size of 228
new rabbit antibody sequences confirmed that rabbit LCDR3s are on average four to five amino acid
residues longer than human and mouse LCDR3s,
which have an arithmetic mean length of 6.5 and
6, respectively.31 With respect to HCDR3 lengths
(Table 2), we found that rabbit antibodies have a
wider range (3 – 22 amino acid residues) than
reported in previous studies that were based on
smaller sample sizes32,33 and, in this regard, are
more closely related to human antibodies (2 –26
amino acid residues) than mouse antibodies (2 –19
amino acid residues). In addition, rabbit and
human antibodies are more closely related in
terms of arithmetic mean ^ SD HCDR3 length,
which is 13.1(^ 4.4) for human HCDR3 (n ¼ 177),32
9.4(^ 2.8) for mouse HCDR3 (n ¼ 1004),32 and
11.6(^ 3.1) for rabbit HCDR3 (n ¼ 228) (Table 2).
On the basis of skewness and kurtosis, our rabbit
HCDR3 lengths distribution practically conforms
to a normal distribution, as do human and mouse
HCDR3 length distributions (Table 2). These
normal distributions of HCDR3 lengths suggested
that neither bacterial nor hybridoma antibody
expression impose an inherent bias. In addition,
they permitted us to validate the hypothesis about
the relative order of the HCDR3 lengths from
these three species by z-test analysis. At the 99 %
confidence level, the HCDR3 length was found
to be in the order human . rabbit . mouse
( p ¼ 0.0001).
A summary of the sequenced VL coding regions
from each of the 12 libraries is given in Table 3. As
expected, most of the VL sequences in all three
immune repertoires were Vk with a higher percentage of Vl sequences in Basilea mutant rabbits (22%)
than in NZW (13%) and b9 wild-type rabbits (11%).
Notably, 68% of Vk sequences in NZW rabbits but
only 15% and 11% in Basilea mutant and b9 wildtype rabbits, respectively, encoded a cysteine at
position 80. Thus, as anticipated, chimeric rabbit/
human Fab libraries derived from Basilea mutant
and b9 wild-type rabbit immune repertoires contain a much lower percentage of clones that display
a free thiol group resulting from an unpaired
cysteine 80. One goal of this study was to determine whether this difference in sequence translates
into a difference in selectable diversity. Whereas
the unselected libraries from the three immune
repertoires were found to have distinct features,
comparison of libraries derived from spleen with
libraries derived from bone marrow revealed only
slight differences. For example, we found a higher
percentage of cysteine 80 encoding Vk sequences
in bone marrow than in spleen from Basilea
mutant and b9 wild-type rabbits (Table 3). Some
of the VH and VL sequences that were independently recovered from bone marrow and spleen of
individual rabbits were clonally related (data not
shown). In summary, all three rabbit immune
repertoires yielded a great diversity of functional
chimeric rabbit/human Fab. This diversity was
found in libraries from both spleen and bone marrow of individual rabbits. Thus, both organs are
excellent sources for the generation of antibody
libraries from immune rabbits.
Library selection and initial analysis of
selected clones
The chimeric rabbit/human Fab libraries were
selected by panning against immobilized Tie-2/
Table 3. Light chain diversity of chimeric rabbit/human
Fab libraries
Vk
(%)
Vk Cys80
(% of Vk)
Total sequenced
16 (89)
15 (88)
14 (78)
17 (94)
62 (87)
11 (69)
9 (60)
12 (86)
10 (59)
42 (68)
18
17
18
18
71 (100)
Basilea mutant libraries
1Ss
6 (32)
13 (68)
2Ss
3 (16)
16 (84)
1Bs
4 (21)
15 (79)
2Bs
4 (20)
16 (80)
Total (%)
17 (22)
60 (78)
2 (15)
0 (0)
3 (20)
4 (25)
9 (15)
19
19
19
20
77 (100)
b9 wild-type libraries
1Sb
4 (20)
2Sb
2 (10)
1Bb
1 (5)
2Bb
2 (10)
Total (%)
9 (11)
2 (12)
1 (6)
2 (10)
3 (17)
8 (11)
20
20
20
20
80 (100)
Vl
(%)
NZW libraries
1S
2 (11)
2S
2 (12)
1B
4 (22)
2B
1 (6)
Total (%)
9 (13)
16 (80)
18 (90)
19 (95)
18 (90)
71 (89)
330
Rabbit Antibody Libraries
Table 4. Selected chimeric rabbit/human Fab
Clone identity
Library origin
Binding propertiesa
Selected (sequenced)
mTie-2
Tie-2
ang-2
Tie-2/ang-2
Tie-1
IgG
BSA
NZW
1S05
2S03
2B01
2B15
1S
2S
2B
2B
4 (4)
2 (2)
22 (5)b
2 (2)
2
þ
2
2
þ
þ
2
þ
2
2
þ
2
þ
þ
þ
þ
2
2
2
2
2
2
2
2
2
2
2
2
Basilea
1S01s
1S02s
1S03s
1B01s
1B03s
1B08s
1B10s
1Ss
1Ss
1Ss
1Bs
1Bs
1Bs
1Bs
2 (1)
9 (2)
5 (1)
2 (1)
2 (1)
2 (1)
1 (1)
2
2
2
2
2
2
2
þ
þ
þ
þ
þ
þ
2
2
2
2
2
2
2
þ
þ
þ
þ
þ
þ
þ
þ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
b9
1S01b
1S02b
1S10b
1S03b
1S06b
1S09b
1B01b
1B03b
1B10b
2S01b
2S08b
1B02b
2B01b
2B02b
2B05b
1Sb
1Sb
1Sb
1Sb
1Sb
1Sb
1Bb
1Bb
1Bb
2Sb
2Sb
1Sb
2Bb
2Bb
2Bb
4 (3)
1 (1)
1 (1)
1 (1)
2 (1)
1 (1)
1 (1)
7 (3)
1 (1)
9 (1)
1 (1)
1 (1)
2 (1)
5 (3)
4 (2)
þ
þ
þ
2
þ
2
2
2
2
þ
þ
2
2
2
2
þ
þ
þ
2
þ
þ
þ
þ
þ
þ
þ
2
þ
þ
þ
2
2
2
þ
2
2
2
2
2
2
2
þ
2
2
2
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Clonally related chimeric rabbit/human Fab that share an amino acid sequence identity of .95% in their variable Ig domains are
assembled in four groups indicated by italics.
a
Binding properties of chimeric rabbit/human Fab in supernatants of IPTG-induced clones selected from NZW (top), Basilea
mutant (center), and b9 wild-type (bottom) rabbit antibody libraries towards different antigens as detected by ELISA; þ, binding;
2, no binding; mTie-2, mouse Tie-2.
b
Unsequenced clones were considered identical by DNA fingerprinting.
ang-2 complex. To avoid the selection of antibodies
to the Fc part of recombinant Tie-2, the selection
was carried out in the presence of 2.5 mg ml21
human IgG. After four rounds of panning, clones
were analyzed for binding to Tie-2/ang-2 complex
by ELISA using a rat anti-HA mAb conjugated to
horseradish peroxidase for detection. The highest
percentage of positive clones was obtained from
b9 wild-type rabbit antibody libraries (97.5%), followed by Basilea mutant (65%) and NZW (45%)
rabbit antibody libraries. Positive clones were
further analyzed by DNA fingerprinting using the
restriction enzyme AluI. Among 30 positive clones
from the NZW rabbit antibody libraries, four distinct fingerprints were identified (Table 4). By contrast, the Basilea mutant rabbit antibody libraries
yielded seven distinct fingerprints among 23 positive clones and the b9 wild-type rabbit antibody
libraries yielded 15 distinct fingerprints among 37
positive clones (Table 4). All 26 positive clones
with distinct fingerprints were subsequently
analyzed by DNA sequencing. To confirm their
identity, some positive clones with identical fingerprints were also sequenced. Four out of four NZW
rabbit clones, two out of seven Basilea mutant
rabbit clones, and nine out of 16 b9 wild-type
rabbit clones revealed unique sequences that were
clonally unrelated. Although all selected clones
bound to Tie-2/ang-2 complex, clonally unrelated
chimeric rabbit/human Fab often revealed distinct
specificities (Table 4). For example, the related
clones 1S01s, 1S02s, 1S03s, 1B01s, 1B03s, and
1B08s, which were independently derived from
spleen and bone marrow of an individual Basilea
mutant rabbit, all bound to human Tie-2 with
similar ELISA signals after normalization for
expression (data not shown). By contrast, the
clonally unrelated Basilea mutant rabbit clone
1B10s bound to human ang-2. Other ang-2 binders
were identified among the NZW rabbit clones
(2B01) and among the b9 wild-type rabbit clones
(clonally unrelated 1S03b and 1B02b). The NZW
rabbit antibody library yielded one and the b9
wild-type rabbit antibody library yielded at least
four distinct chimeric rabbit/human Fab that
recognized both human and mouse Tie-2 (Table 4).
All the selected clones bound to one of the two
components of the Tie-2/ang-2 complex; none was
found to exclusively recognize the Tie-2/ang-2
complex. Among the selected chimeric rabbit/
human Fab only two clones (1S05 and 2S03) contained a lambda light chain and only one clone
331
Rabbit Antibody Libraries
(2B15) contained a kappa light chain with cysteine
80. Most notably, all three of these clones were
derived from the NZW rabbit antibody libraries,
which yielded only one clone that contained a
kappa light chain without cysteine 80. By contrast,
all clones selected from the Basilea mutant and b9
wild-type rabbit antibody libraries contained
kappa light chains without cysteine 80.
Further analysis of selected clones
For further analysis, an assortment of selected
chimeric rabbit/human Fab were produced as
soluble Fab in E. coli and purified by affinity chromatography using goat anti-human F(ab0 )2 NHS
resin columns. The selected chimeric rabbit/
human Fab that bound Tie-2 were analyzed for
binding to human umbilical vein-derived endothelial cells (HUVEC) by flow cytometry. All
clones, including those that recognized both
human and mouse Tie-2, were found to bind to
HUVEC (Figure 4). Thus, the various Tie-2 epitopes recognized by the selected chimeric rabbit/
human Fab are displayed by native Tie-2 expressed
on the cell surface and are accessible targets for
antiangiogenic therapy. To compare clones derived
from different immune repertoires, we measured
their affinities to human Tie-2 by surface plasmon
resonance. We focused on unrelated clones that
gave the strongest ELISA signals after normalization for expression. Representing the NZW rabbit
immune repertoire, chimeric rabbit/human Fab
1S05, which contains a lambda light chain, and
2B15, which contains a kappa light chain with
cysteine 80, revealed a monovalent affinity of
15 nM and 12 nM, respectively (Figure 5; Table 5),
confirming our previous result that chimeric
rabbit/human Fab with a cysteine 80 in their
kappa light chain can still have reasonable
affinities.15 However, chimeric rabbit/human Fab
1S02s representing the Basilea mutant rabbit and
1S09b representing the b9 wild-type rabbit immune
repertoire revealed dramatically stronger affinities
in the 400 –500 pM range (Table 5). As obvious
from the Biacore sensorgrams shown in Figure 5,
these stronger affinities arose from much lower
dissociation rate constants (Table 5), which likely
are a result of affinity maturation in vivo.34
Figure 4. Analysis of selected chimeric rabbit/human
Fab by flow cytometry. For indirect immunofluorescence
staining, HUVEC were incubated with purified chimeric
rabbit/human Fab followed by FITC-conjugated secondary antibodies. (a) Shown is the mean fluorescence
intensity (MFI) after subtracting the background of
FITC-conjugated secondary antibodies (n ¼ 3). (b) Flow
cytometry histogram showing the binding of chimeric
rabbit/human Fab 1S02s to HUVEC as bold line. The
background of FITC-conjugated secondary antibodies is
shown as a broken line. Chimeric rabbit/human Fab
directed to human A33 antigen14 were used as negative
control (thin line). The y axis gives the number of events
in linear scale, the x axis the fluorescence intensity in
logarithmic scale.
Cross-reactivity with human and mouse antigen
A key motivation for the generation of therapeutic mAbs from rabbits is their potential crossreactivity with human, non-human primate, and
mouse antigens, a highly relevant property facilitating preclinical evaluation.3 Supporting this
claim, we here describe for the first time a rabbit
mAb that recognizes both human and mouse antigen with the same affinity. As revealed by ELISA,
NZW clone 2S03 and b9 wild-type clones 1S06b,
2S01b, 2S08b, as well as a group of related b9
wild-type clones comprising 1S01b, 1S02b, and
1S10b were found to bind both human and mouse
Table 5. Binding parameters of selected chimeric rabbit/
human Fab directed to human Tie-2
Fab
1S05
2B15
1S02s
1S09b
kon/104 (M21 s21)
koff/1024 (s21)
Kd (nM)
13
8.4
7.5
7.3
20
10
0.29
0.39
15
12
0.39
0.53
Association (kon) and dissociation (koff) rate constants were
determined using surface plasmon resonance. Human Tie-2
was immobilized on the sensor chip. The dissociation constant
(Kd) was calculated from koff/kon.
332
Rabbit Antibody Libraries
Figure 5. Analysis of selected chimeric rabbit/human Fab by surface plasmon resonance. Shown are Biacore sensorgrams obtained for the binding of chimeric rabbit/human Fab 1S05 (NZW), 2B15 (NZW), 1S02s (Basilea mutant), and
1S09b (b9 wild-type) to immobilized human Tie-2. For association, Fab were injected at five different concentrations
(150, 125, 100, 75, and 50 nM; top to bottom) between t ¼ 125 seconds and t ¼ 370 seconds using a flow rate of
10 ml min21. For dissociation, the flow rate was increased to 50 ml min21. RU, resonance units.
Tie-2 (Table 4), whose amino acid sequences are
93% identical. The cross-reactivity of chimeric rabbit/human Fab 2S08b, which had already been
shown to bind to a native epitope (Figure 4), was
analyzed quantitatively by surface plasmon resonance. As detailed in Table 6, 2S08b bound with a
monovalent affinity of approximately 1 nM to
both human and mouse Tie-2. Thus, 2S08b binds
to an epitope on Tie-2 that is conserved between
human and mouse but does not overlap with the
ang-2 binding site.
Conclusions
We compared three different rabbit immune
repertoires with respect to the generation and
selection of phage libraries displaying chimeric
rabbit/human Fab, an intermediate format for antibody humanization. All three rabbit immune
repertoires yielded a great diversity of chimeric
rabbit/human Fab whether derived from spleen
or bone marrow. However, significant differences
were found after the libraries had been subjected
to a stringent selection over four rounds of
panning. Compared to the commonly used NZW
Table 6. Cross-reactivity of chimeric rabbit/human Fab
2S08b with human and mouse Tie-2
Antigen
kon/104 (M21 s21)
koff/1024 (s21)
Kd (nM)
hTie-2
mTie-2
12.7 ^ 2.4
13.4 ^ 1.5
1.44 ^ 0.19
1.37 ^ 0.25
1.2 ^ 0.2
1.0 ^ 0.1
Association (kon) and dissociation (koff) rate constants were
determined using surface plasmon resonance. Human or
mouse Tie-2 was immobilized on the sensor chip. The dissociation constant (Kd) was calculated from koff/kon. hTie-2,
human Tie-2; mTie-2, mouse Tie-2.
rabbit immune repertoire, the Basilea mutant
rabbit immune repertoire and, in particular, its parental b9 wild-type immune repertoire yielded (i) a
greater percentage of selected clones that bound to
the antigen, (ii) a greater diversity among the
selected clones with respect to DNA sequence and
in the b9 wild-type rabbit immune repertoire,
diversity of antigen specificity, and (iii) selected
clones with much stronger affinity to the antigen.
The advantages of the Basilea mutant and b9
wild-type rabbit immune repertoires over the
NZW immune repertoire correlated inversely with
the frequency of kappa light chains containing a
cysteine 80 in the unselected libraries. Although
68% of Vk sequences in unselected libraries from
NZW rabbits were found to contain a cysteine 80,
only one out of four selected NZW clones contained a kappa light chain with cysteine 80. On
the basis of two different antigens, the same ratio
was found previously.14,15 Thus, whereas a majority
of antibodies in immune NZW rabbits contains
kappa light chains with a disulfide bridge between
cysteine 80 and cysteine 171, a much lower percentage of chimeric rabbit/human Fab containing a
kappa light chain with cysteine 80 has been
selected. This finding makes it likely that an
unpaired cysteine 80 reduces the selectable antibody diversity. On the other hand, a higher selectable antibody diversity is the likely explanation
for the fact that Basilea mutant and b9 wild-type
rabbit immune repertoires with higher levels of
expression of Vk sequences lacking cysteine 80
yield chimeric rabbit/human Fab with superior
properties. The fact that the HCDR3 length distribution in rabbit antibodies is more similar to
human than mouse antibodies is highly relevant
for the generation of therapeutic mAbs from
rabbit immune repertoires, since this region is conserved in the process of antibody humanization.
333
Rabbit Antibody Libraries
As a consequence, humanized rabbit antibodies
may be more closely related to human antibodies
than humanized mouse antibodies.
Materials and Methods
Reagents
Lyophilized recombinant human and mouse Tie-2/Fc
fusion proteins (330 kDa), which contain the extracellular
domain of human or mouse Tie-2 fused to human IgG1
Fc via a polypeptide linker, and recombinant human
ang-2 (66 kDa) were purchased from R & D Systems
(Minneapolis, MN). Horseradish peroxidase-conjugated
goat anti-rabbit Fc polyclonal antibodies were from
Jackson ImmunoResearch Laboratories (West Grove,
PA). Goat anti-human Fab polyclonal antibodies were
from Bethyl Laboratories (Montgomery, TX). Goat antihuman kappa light chain polyclonal antibodies were
from Pierce (Rockford, IL). Horseradish peroxidaseconjugated rat anti-HA mAb 3F10 was from Roche
Molecular Biochemicals (Mannheim, Germany). FITCconjugated goat anti-human kappa light chain polyclonal
antibodies were from Southern Biotechnology Associates
(Birmingham, AL). Human umbilical vein-derived
endothelial cells (HUVEC) were purchased from BioWhittaker (Walkersville, MD) and maintained in EGM
complete medium supplemented with bovine brain
extract (BioWhittaker).
Immunization
Two rabbits from the NZW laboratory strain with allotypes a3/a3 b4/b4 and a1/a3 b4/b4, two rabbits from
the Basilea mutant strain with allotypes a1/a3 bas/bas
and a1/a2 bas/bas, and two b9 wild-type rabbits with
allotype a1/a2 b9/b9 were chosen for immunization.
The b9 wild-type rabbits represent the parental strain
from which the Basilea mutation arose.35 All allotypes
were confirmed by serotyping using anti-a1, a2, a3, b4,
b5, b6, b9, and bas antisera as described.36 Each rabbit
received an initial immunization with a complex of equimolar amounts of human Tie-2 and ang-2 (12.5 mg Tie-2
and 5 mg ang-2) that had been incubated at 37 8C for 30
minutes immediately before emulsification with Ribi
adjuvant (MPL þ TDM þ CWS in PBS) according to the
manufacturer’s
instructions
(Ribi
Immunochem
Research, Hamilton. MT). A total of 1 ml was distributed
in four subcutaneous sites on the back. After the initial
immunization, three identical additional boosts were
given at three-week intervals. Antisera from immune
rabbits were analyzed for binding to the immunogens
by ELISA using horseradish peroxidase-conjugated goat
anti-rabbit Fcg polyclonal antibodies.
amplified from first strand cDNA and fused to human
Ck and CH1 encoding sequences, respectively, followed
by assembly of chimeric rabbit/human light chain and
Fd fragment encoding sequences and by asymmetric
Sfi I cloning into phagemid vector pComb3X. Note that
the reverse primers that hybridize to the Jk region for
amplification of rabbit Vk encoding sequences eliminate
the b9 wild-type cysteine at position 108. The resulting
chimeric rabbit/human Fab libraries were designated
1S, 2S, 1B, and 2B (NZW rabbits); 1Ss, 2Ss, 1Bs, and 2Bs
(Basilea mutant rabbits); and 1Sb, 2Sb, 1Bb, and 2Bb (b9
wild-type rabbits). For validation, approximately 20
IPTG-induced clones from each unselected library were
analyzed for the expression of chimeric rabbit/human
Fab by ELISA using goat anti-human IgG and goat antihuman kappa light chain polyclonal antibodies for capture and a rat anti-HA mAb (an epitope tag from
pComb3X) conjugated to horseradish peroxidase for
detection. Clones that gave a signal at least fourfold
over background were defined as positive and further
analyzed by DNA sequencing. Statistics (median,
mean ^ SD, skewness, kurtosis, and z-test) were calculated using Microsoft Excel software. All 12 libraries
were panned in parallel against Tie-2/ang-2 complex
immobilized on Costar 3690 96-well ELISA plates
(Corning; Acton, MA). Four rounds of panning14,30 were
carried out using 700 ng of Tie-2/ang-2 complex in the
first round, 350 ng in the second round, and 140 ng
Tie-2/ang-2 complex in the third and fourth rounds. To
eliminate the selection of clones that bind to the human
IgG1 Fc part of recombinant human Tie-2/Fc fusion protein, 2.5 mg ml21 human IgG (Pierce, Rockford, IL) were
added to the phage preparations during selection. After
the final round of panning, approximately ten IPTGinduced clones from each library were analyzed for
binding to 100 ng immobilized Tie-2/ang-2 complex,
Tie-2, ang-2, human Tie-1/Fc fusion protein (R & D
Systems), human IgG, and BSA by ELISA using a rat
anti-HA mAb conjugated to horseradish peroxidase for
detection. Clones that bound Tie-2/ang-2 complex,
Tie-2, or ang-2 were further analyzed by DNA fingerprinting and sequencing.
DNA fingerprinting and sequencing
For DNA fingerprinting, Fab encoding inserts in
pComb3X were amplified by PCR, using the primers
GBACK (50 -GCC CCC TTA TTA GCG TTT GCC ATC-30 )
and OMPSEQ GTG (50 -AAG ACA GCT ATC GCG ATT
GCA GTG-30 ) and digested with Alu I, a frequently cutting restriction enzyme with recognition sequence AG/
CT (Promega, Madison, WI). The restriction fragments
were separated in 4% (w/v) agarose gels and stained
with ethidium bromide. Primers NEWPELSEQ and
OMPSEQ14 were used for DNA sequencing of rabbit VH
and VL encoding regions, respectively, from purified
phagemid DNA.
Library generation and selection
Five days after the final boost, spleen and bone
marrow from both femurs of the immune rabbits were
harvested separately and used for total RNA preparation
and first strand cDNA synthesis as described.14,30 Twelve
separate libraries representing immune repertoires
derived from spleen or bone marrow of individual
rabbits were generated. Detailed protocols for the generation of chimeric rabbit/human Fab libraries in the
phagemid vector pComb3X are published elsewhere.14,30
In brief, rabbit Vk, Vl, and VH encoding sequences were
Protein expression and purification
Soluble Fab were expressed from gene III fragmentdepleted phagemid vector pComb3X and purified using
goat anti-human F(ab0 )2 NHS resin columns as described.30
Flow cytometry
HUVEC were washed with Hepes buffered saline
solution (HBSS; BioWhittaker) detached by mild
334
trypsinization with 0.025% trypsin, 0.01% EDTA in HBSS
(BioWhittaker), washed with PBS, and resuspended at a
concentration of 106 cells ml21 in flow cytometry buffer
(1% (w/v) BSA, 0.03% (w/v) NaN3, 25 mM Hepes in
PBS, pH 7.4). Aliquots of 100 ml containing 105 cells
were distributed into wells of a V-bottom 96-well plate
(Corning) for indirect immunofluorescence staining
using 5 mg ml21 purified rabbit/human Fab and a 1:100
dilution of FITC-conjugated goat anti-human kappa
light chain polyclonal antibodies in flow cytometry
buffer. Incubation with primary and secondary antibodies was for 40 minutes at room temperature. Flow
cytometry was performed using a FACScan instrument
from Becton-Dickinson (Franklin Lakes, NJ).
Surface plasmon resonance
Surface plasmon resonance for the determination of
association (kon) and dissociation (koff) rate constants for
the interaction of chimeric rabbit/human Fab with Tie-2
was performed on a Biacore instrument (Biacore AB,
Uppsala, Sweden). A CM5 sensor chip (Biacore AB) was
activated for immobilization with N-hydroxysuccinimide and N-ethyl-N0 -(3-dimethylaminopropyl)carbodiimide according to the methods outlined by the
supplier. Recombinant human or mouse Tie-2/Fc fusion
protein was coupled at a low density (500 – 1000 resonance units) to the surface by injection of 5 – 10 ml of a
10 ng/ml sample in 20 mM sodium acetate (pH 3.5).
Subsequently, the sensor chip was deactivated with 1 M
ethanolamine hydrochloride (pH 8.5). Binding of
chimeric rabbit/human Fab to immobilized human or
mouse Tie-2 was studied by injection of Fab at five
different concentrations ranging from 40 to 150 nM. PBS
was used as the running buffer. The sensor chip was
regenerated with 20 mM HCl and remained active for at
least 20 measurements. The kon and koff values were calculated using Biacore AB evaluation software. The equilibrium dissociation constant Kd was calculated from
koff/kon. Data obtained from different sensor chips
revealed a high consistency.
Acknowledgements
We thank Marikka Elia and Glendowlyn Cooper
for excellent technical assistance and Drs Michael
G. Mage, David H. Margulies, David J. Segal, and
Guibin Yang for help and discussion. This work
was supported by an Investigator Award from the
Cancer Research Institute (to C.R.) and by National
Institutes of Health Grants AI 37470 (to C.F.B.III)
and CA 94966 (to C.R.).
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Edited by J. Karn
(Received 13 September 2002; received in revised form 25 October 2002; accepted 29 October 2002)